Respiratory complex I is the first electron-acceptor in the bioenergetic chains of many organisms. This protein complex transfers two electrons from NADH to quinone (Q), and transfers the free energy of this reaction to pumping of four protons. The proton electrochemical gradient across the bioenergetic membrane drives the synthesis of ATP. The ~1 MDa mitochondrial complex I, which comprises ca. 45 different protein subunits, is at the centerstage of mitochondrial dysfunction and diseases, and plays a key role in NADH recycling and ROS production. Despite extensive structural and biochemical investigations, the molecular mechanism of complex I remains unknown. By performing free energy calculations and microseconds of molecular dynamics (MD) simulations on small models of bacterial and mitochondrial complexes, we have identified quinone binding sites in the unique ~35 Å quinone channel of complex I. The recent high resolution cryo EM data provided support to these computational findings. By combining site directed mutagenesis with classical MD simulation approaches, we identified how point mutations perturb the subtle substrate-protein dynamics. Hybrid quantum mechanical/molecular mechanical MD simulations were employed to study the transfer of <i>pumped</i> and <i>substrate</i> protons, based on which we redefine the role of supernumerary subunits of respiratory complex I. Our joint approach utilizing multiscale molecular simulations, biochemistry and high-resolution structural data, yield detailed mechanistic insights into the redox-coupled proton pumping by complex I.

Redox-coupled protonation and conformational dynamics in respiratory complex I

ACS Fall 2021

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